RU2491961C2 - Artificial dura mater and method of its production - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to chemical-pharmaceutical industry and represents artificial dura mater, produced from electrospinning layers by technology of electorspinning, with electrospinning layer, consisting of, at least, hydrophobic electrospining layer, which is produced from one or several hydrophobic polymers, selected from polylatic acid and polycaprolactone.

EFFECT: invention ensures creation of artificial dura mater, which has good tissue compatibility, anti-adhesiveness and possibility of introducing medications, preventing cerebrospinal fluid outflow during regeneration of person's own dura mater.

30 cl, 7 ex, 11 dwg

Description

The technical field of the invention

The invention is directed to the study of artificial biofilms, namely artificial dura mater, and methods for their production.

State of the art

Damage to the dura mater is a common problem in neurosurgery. Open craniocerebral injuries (obtained at work, as a result of an accident or military action), tumor invasion, congenital damage to the meninges or other diseases of the skull can lead to damage to the dura mater. Such damage to the dura mater requires timely treatment in order to prevent the outflow of cerebrospinal fluid, the formation of a hernia of the brain and stress from exposure to atmospheric pressure. Otherwise, it can be life threatening.

Currently, despite the fact that there are a large number of substitutes for the dura mater, the materials used in the substitutes can be divided into four types: autologous fascia, allograft, natural or artificial substance, and xenograft. However, the use of these materials in medical practice inevitably leads to problems such as a high level of infections. According to statistics, the incidence of craniotomy is 4%; the dura mater made from the mucous membrane of the porcine small intestine gives an infection rate of 3.4%; and the dura mater made from collagen is an infection rate of 3.8%. Due to the blood-brain barrier, as soon as intracranial infection occurs, it will not be possible to provide the required concentration of anti-infective drugs in the brain plasma, and the infection will be difficult to control. Also, existing artificial substitutes for the dura mater do not have the ability to deliver drugs to the pia mater. Consequently, postoperative infection control is often not effective.

Moreover, one of the main reasons for the need to restore the dura mater using a dura graft is damage to the meninges due to tumor invasion. More than half of the cases of brain tumors cannot be completely removed surgically and therefore chemotherapy is necessary after surgery. Many chemotherapeutic drugs are toxic and cannot overcome the blood-brain barrier, so it is impossible to achieve an effective concentration of drugs, which leads to a decrease in the results of chemotherapy.

Existing artificial substitutes for the dura mater usually do not contain drugs of interest. For example, due to the dense structure, it is impossible to ensure the introduction of drugs into the autologous fascia in a natural way, it is also difficult to introduce drugs into an allograft or xenograft. However, substitutes for the dura mater based on synthetic material, due to their good elasticity, can be easily filled with drugs. On the other hand, due to the limitation of the methods of drug administration, it is also not easy to introduce the drug into the artificial dura mater, as well as to provide the possibility of drug release after transplantation to achieve therapeutic goals. To date, the main method of introducing an anti-infective drug into an artificial substitute for the dura mater is by impregnating the substitute with the drug. When using this method, most of the drug remains on the surface of the artificial dura mater, while it is difficult to control, which makes it difficult to achieve controlled release.

In conclusion, it should be noted that the existing artificial substitutes for the dura mater have such disadvantages as high infection rates, low biocompatibility, incomplete absorption, difficulty in administering the drug and controlling its effective release.

Description of the invention

To solve at least one of the technical problems described above, according to the present invention, an artificial dura mater is provided, which is distinguished by excellent tissue compatibility, perfect release, full absorption, good mechanical properties, low infections and the possibility of administering drugs.

In addition, a method for manufacturing artificial dura mater is provided.

According to the invention, the artificial dura mater consists of electro-spun layers. The aforementioned electrospun layers consist of at least one hydrophobic electrospun layer, which is made from one or more hydrophobic polymers by electrospinning. The above hydrophobic polymer is selected from the group consisting of hydrophobic aliphatic polyester, polyester ether copolymer, polyorthoester, polyurethane, polyanhydride, polyphosphazene, polyamino acid, copolymers, as well as mixtures thereof.

Also, a hydrophobic aliphatic polyester is at least one substance selected from the group consisting of polylactic acid, polyglycolide, polycaprolactone and polyhydroxybutyrate (PHB). The aforementioned polyester-ether copolymer is at least one substance selected from the group consisting of polydioxanone (PDO), a glycol-lactic acid copolymer and a polybutylene terephthalate / glycol copolymer. The aforementioned polyanhydride is at least one substance selected from the group consisting of polysebacic acid polyanhydride (PSPA) -tetanoic acid anhydride, type-I polyanhydride, type-II polyanhydride, type-III polyanhydride and type-IV polyanhydride.

The artificial dura mater, consisting of a hydrophobic layer, has a strength similar to that of the human dura mater. It can seal brain injuries and prevent the flow of cerebrospinal fluid during the regeneration of the human dura mater. The hydrophobic layer is unfavorable for the movement and binding of cells, whereby the goal of release can be achieved. In fact, several hydrophobic layers can be used in order to meet different strength requirements.

In addition, according to the invention, in the artificial dura mater, at least one hydrophilic electro-spun layer can be placed on the aforementioned hydrophobic electro-spun layer. The aforementioned hydrophilic layer was prepared by electrospinning using one or more types of hydrophilic polymers selected from the group consisting of chondroitin sulfate, heparin, agar, glucan, algin, cellulose, modified cellulose, sodium alginate, starch, gelatin, fibrinogen, silk protein, polymer peptide imitating elastin, collagen, chitosan, modified chitosan, hydrophilic polyurethane, polyethylene glycol, polymethyl methacrylate, polymethyl methacrylate, hydroxybutyrate co-hydroxyvalerate , RNVNNh (polyhydroxybutyrate-co-hydroxyhexanoate), polyvinyl alcohol, polylactide and mixtures thereof.

During transplantation of the dura mater into the brain, the hydrophobic layer, which plays the role of a release agent, is located close to the surface of the brain, while the hydrophilic layer, which provides a thin nanofiber scaffold for adhesion, movement, growth, and differentiation of cells, is located far from the brain. Since the hydrophilic layer is made of hydrophilic materials of good biocompatibility, it can significantly enhance the processes of movement and proliferation of stem cells and fibroblasts, and, therefore, contribute to the growth of autologous dura mater. In fact, several hydrophobic layers can be used in order to meet different requirements.

According to one embodiment of the invention, the inventive dura may also have a transition layer between the hydrophobic and hydrophilic layers. The transition layer is made by electrospinning using one or more polymers, and it has hydrophilicity, which gradually increases from the side closest to the hydrophobic electrospinning layer, to the side closest to the hydrophilic electrospinning layer. The presence of a transition layer can improve hydrophilicity between the hydrophobic and hydrophilic layers.

According to another embodiment of the invention, each of the hydrophobic, hydrophilic as well as transition layers can be mixed with a cytokine and / or drug. Miscible layers can provide release of the cytokine and / or drug into the brain during transplantation, dressing with absorption of the polymer scaffold. Then the cytokine and / or drug can achieve the goal of preventing local infection, adhesion and / or relapse of the tumor or to help restore the autologous dura mater.

According to another embodiment of the invention, the adhesion of the hydrophobic and hydrophilic layers can be achieved using a cytokine and / or drug.

The cytokine mentioned in the invention is a factor that plays an important role in the adhesion, movement, proliferation and differentiation of fibroblasts, which is selected from the group consisting of interleukin, colony stimulating factor, tumor necrosis factor, platelet growth factor, main fibroblast growth factor, and combinations thereof . Cytokine can speed up the process of repairing damaged dura mater.

A medicament referred to in the invention is one or more substances selected from the group consisting of antibiotics, hemostatic agents, release agents and anti-tumor drugs. These drugs are placed on artificial dura mater according to actual need. After transplantation of the artificial dura mater, drugs are gradually released so as to ensure that the therapeutic goal is met during the breakdown of the polymers and the regeneration of the damaged dura mater. Thus, the blood-brain barrier to the drug is overcome.

In addition, the cytokine and / or drug can be enclosed in a hydrogel. Due to the effects of adhesion and fixation of the hydrogel in accordance with the position in place, the drug can be released in equal portions or in a special way. The aforementioned hydrogel consists of one or more substances selected from the group consisting of a polysaccharide polymer, a polypeptide polymer and a synthetic hydrophilic high molecular weight polymer.

According to one embodiment of the invention, the hydrophobic electro-spun layer of the aforementioned artificial dura mater consists of fibers with a diameter of 50-1000 nm. According to one embodiment of the invention, the hydrophobic electro-spun layer consists of pores whose size is less than 3 microns, while the hydrophilic electro-spun layer consists of fibers with a diameter of 5-200 microns and a pore size of 20-200 microns. The pore size is largely dependent on the diameter of the fiber. If the fiber diameter decreases, the pore size also decreases. Thus, controlling the diameter of the fiber can control the pore size of the electro-spun layer. The average diameter of human cells is 10-50 microns. The pia mater mainly consists of fibroblasts and collagen fibers secreted from fibroblasts. Most fibroblasts have a diameter of 20-30 microns. The pore size of the hydrophobic layer is less than 3 μm, which can prevent cell penetration and adhesion between the dura mater and brain tissue. The hydrophilic layer consists of pores whose size is equal to or larger than the average diameter of the cell, which can facilitate the penetration and movement of the cell.

According to another aspect of the invention, a method for producing artificial dura mater also includes the following steps:

A) dissolving a hydrophobic polymer in a solvent in order to obtain a hydrophobic electrospinning solution, in which the aforementioned hydrophobic polymer is selected from the group consisting of hydrophobic aliphatic polyester, polyester-ether copolymer, polyorthoester, polyurethane, polyanhydride, polyphosphazene, polyamino acids, and their polyamino acids mixtures;

B) fabricating by electrospinning a hydrophobic electrospinning layer such as a film (or web type) from the aforementioned hydrophobic electrospinning solution, thereby manufacturing the aforementioned artificial dura mater.

In addition, the aforementioned hydrophobic aliphatic polyester is at least one substance selected from the group consisting of polylactic acid, polyglycolide, polycaprolactone and polyhydroxybutyrate. The aforementioned polyester-ether copolymer is at least one substance selected from the group consisting of polydioxanone (PDO), a glycol / lactic acid copolymer and a terephthalic acid / glycol polybutylene terephthalate copolymer. The aforementioned polyanhydride is at least one substance selected from the group consisting of polysebacic acid-cetane bisacid anhydride polyanhydride, type I polyanhydride, type II polyanhydride, type III polyanhydride and type IV polyanhydride.

The method applicable to the principle of electrospinning creates an artificial dura mater using a specific polymer. Dura can effectively prevent its adhesion to brain tissue.

To further prevent adhesion of the artificial dura mater to the brain tissue, it is necessary to control the diameter of the fibers, and as a result, the pore size of the scaffold is also controlled. In this way, the movement of cells can be stopped. The diameter of the fibers of the hydrophobic electro-spun layer can be controlled in the range of 50-1000 nm. According to one embodiment of the invention, the pore size of the hydrophobic electro-spun layer is less than 3 microns.

In the method described in paragraph B) above, the aforementioned electrospinning is performed using a micro-injection pump operating at a rate of 0.1-5.0 ml / h and a high voltage generator operating at a voltage of 5-40 kV and a feed distance of 5, 0-30.0 cm

To achieve a better effect in clinical therapy, the invention also provides a method similar to electrospinning for creating a hydrophilic electrospinning layer on the aforementioned hydrophobic layer, which comprises the following steps:

a ') Dissolution of the hydrophilic polymer in a solvent to obtain a hydrophilic electrospinning solution in which the aforementioned hydrophilic polymer is selected from the group consisting of chondroitin sulfate, heparin, agar, glucan, algin, modified cellulose, sodium alginate, starch, cellulose, gelatin, fibrinogen , silk protein, polymer-peptide imitating elastin, collagen, chitosan, modified chitosan, hydrophilic polyurethane, polyethylene glycol, polymethyl methacrylate, polymethyl methacrylate, hydro ibutirat co-hydroxyvalerate, RNVNNh (polyhydroxybutyrate-co-hydroxyhexanoate), polyvinylalcohol polylactide.

b ') Placing the aforementioned hydrophilic electrospinning solution by electrospinning on the aforementioned hydrophobic layer to create the aforementioned hydrophilic electrospinning layer.

The hydrophilic electro-spun layer is installed at a great distance from the surface of the brain to facilitate cell movement and regeneration of the dura mater. To facilitate the penetration of cells, the hydrophilic electro-spun layer consists of fibers with a diameter of 5-200 microns and pores, the size of which is 20-200 microns.

In paragraph b '), the creation of a hydrophilic electrospun layer, the aforementioned electrospinning is performed using a micro-injection pump operating at a rate of 0.1-20.0 ml / h and a high voltage generator operating at a voltage of 10-40 kV, and with a feed distance of 5 , 0-30.0 cm.

During electrospinning, usually hydrophobic and hydrophilic polymers must be dissolved in appropriate solvents, respectively, in order to create electrospinning solutions. Often, the aforementioned solvents are volatile organic solvents which include, but are not limited to, methane acid, acetic acid, ethyl alcohol, acetone, dimethylformamide, dimethylacetamide, dihydrofuran, dimethyl sulfoxide, hexafluoroisopropyl alcohol, trifluoroethyl ethyl methane, trifluoroethyl methane alcohol, methyl dichloromethane, methyl dichloromethane alcohol, chloroform, dioxane, trifluoroethane, trifluoroethyl alcohol and mixtures thereof. Volatile organic solvents will quickly evaporate during the process of creating electro-spun layers, while the final electro-spun layers will not or will contain to a small extent residual organic solvent that can be removed at later stages. In some cases, water can be used as a solvent, which can then be removed by heating in an oven or drying naturally after creating electro-spun layers.

In addition, the method for manufacturing artificial dura mater provided by the present invention also includes, prior to manufacturing the aforementioned hydrophilic electrospin layer, creating a transition layer by electrospinning between the aforementioned hydrophilic and hydrophobic layers, the aforementioned transition layer having hydrophilicity, which gradually increases from the side closest to the aforementioned hydrophobic electrospun layer, to the side closest to the aforementioned hydrophilic elec spinning layer. Materials, solvents and electrospinning parameters for the manufacture of the transition layer can be determined based on the actual situation and need. The presence of a transition layer can improve hydrophilicity between the hydrophilic and hydrophobic layers.

In one embodiment, during the creation of each electrospinning layer by electrostatic spinning, cytokine and / or drug can be added to the respective electrospinning solutions. Using the mixing technique, mixed polymer layers and a cytokine and / or drug are better suited for clinical use and have a better therapeutic effect.

The method for manufacturing the artificial dura mater provided in the present invention also includes the distribution of the cytokine and / or drug on the aforementioned hydrophobic electro-spun layer and / or the aforementioned hydrophilic electro-spun layer by bioprinting. Bioprinting is a technology for printing a cytokine and / or drug on bio-paper, consisting of scaffolds of the aforementioned hydrophobic electro-spun layers and / or of the aforementioned hydrophilic electro-spun layers.

In order to evenly distribute the aforementioned cytokine and / or drug and fix them at a certain point on the layers, it is possible to enclose the cytokine and / or drug in a hydrogel.

In particular, the aforementioned bioprinting consists of the following steps:

a '') mixing the hydrogel solution with a cytokine and / or drug to create a solution;

and

b '') printing the aforementioned solution on the aforementioned hydrophobic electro-spun layers and / or hydrophilic electro-spun layers using bioprinting technology.

The above hydrogel solution in the invention consists of aqueous solutions of a polysaccharide polymer, a polypeptide polymer, a synthetic hydrophilic polymer, or mixtures thereof. Moreover, the aforementioned polysaccharide polymer includes, but is not limited to, starch, cellulose, sodium alginate, hyaluronic acid and chitosan. The aforementioned polypeptide polymer includes, but is not limited to, collagen, poly-L-lysine and a lactic and glycolic acid copolymer (PLGA). The aforementioned synthetic hydrophilic polymer includes, but is not limited to, acrylic acid, polymethacrylic acid, polyacrylamide, and N-isopropyl acrylamide.

Under normal conditions, the hydrogel is liquid. At the appropriate temperature or under certain conditions, it can for a short time turn into a gel, while it has good adhesion. According to the invention, some hydrogels require a cross-linking agent to participate in the reaction. Therefore, the method also includes treating the aforementioned hydrophobic and / or hydrophilic layers with a solution containing a cross-linking agent prior to bioprinting. When treated with the aforementioned solution containing a crosslinking agent, the crosslinking agent adheres to the layer or layers. After this, the cytokine and / or drug will be added to the hydrogel solution, and the mixed solution will be placed in the print head. During printing, when a hydrogel solution with a cytokine and / or drug reaches the electro-spun layers, it hardens and adheres to the layers.

With uniform and stable printing, it is possible to ensure uniform release of the cytokine and / or drug. While in individual printing with different speeds and positions in the particular case, it is possible to ensure the release of the cytokine and / or drug in a particular area. The choice of cross-linking agent depends on the type of hydrogel. For example, if the hydrogel is sodium alginate, the cross-linking agent is calcium chloride; if the hydrogel is fibrinogen, the cross-linking agent is thrombin.

Compared with the prior art, the invention has the following advantages:

(1) Its mechanical properties meet the requirements of tensile strength and elasticity, in addition, it is moisture resistant and has anti-adhesive properties.

(2) The materials consisting of shells are non-toxic and harmless to the human body, have good compatibility, provide complete absorption after transplantation, which avoids the appearance of a tumor or cancer.

(3) Animal tissue is not used in the manufacture of the sheath; therefore, risks such as immune rejection, spread of viruses and infection can be avoided.

(4) The provided double layers can prevent adhesion, promote the growth of autologous cells, which, in turn, contribute to the timely restoration of the dura mater.

(5) Using bioprinting technology, therapeutic substances can be introduced into the membrane and released after transplantation in a controlled manner.

(6) The materials presented in large quantities in the document are inexpensive and convenient for transportation and storage.

(7) The production method includes simple procedures, is characterized by low costs and is simple for industrial implementation.

(8) Simple application in medical practice, individual use for the patient is also possible.

Additional aspects and advantages of the invention will be presented in the further description. After reading and putting into practice, some issues will become more apparent.

Brief description of the accompanying drawings

The above and / or additional aspects and advantages or additional aspects and advantages of the present invention will become more apparent and will be easier to understand by reading the following description in conjunction with the drawings and embodiments, in which:

Figure 1 - image of the electrospinning process for the manufacture of artificial dura mater according to the present invention.

Figure 2 - image of the preparation of artificial dura mater according to the present invention, which combines electrospinning and bioprinting.

Figure 3 is an image of an artificial dura mater according to the present invention, consisting of the aforementioned hydrophobic electro-spun layer (s).

Figure 4 is an image of an artificial dura mater according to the present invention, consisting of the aforementioned hydrophobic electro-spun layer and the aforementioned hydrophilic electro-spun layer.

Figure 5 - image of the artificial dura mater according to the present invention, consisting of hydrophobic, hydrophilic and transition layers.

6A is a depiction of the aforementioned mixed artificial dura mater according to the present invention, consisting of a hydrophobic electro-spun layer and a hydrophilic electro-spun layer.

Figure 6B is an enlarged image of a view of I in Figure 6A.

Figure 7A is a depiction of the aforementioned mixed artificial dura mater according to the present invention, consisting of a hydrophobic electro-spun layer, a hydrophilic electro-spun layer and a transition layer.

Figure 7B is an enlarged image of a view of II in Figure 7A.

Figure 8A is an image of the aforementioned artificial dura mater according to the invention, obtained in combination with bioprinting technology.

Figure 8B is an enlarged image of a view of III in Figure 8A.

The positions in the figures mean the following:

1. Electrospinning spray apparatus;

2. Spinning fibers;

3. High voltage power supply;

4. The receiving device;

5. Bioprinting head;

6. The vessel;

7. Hydrophobic spinning fiber;

8. The drug;

9. Hydrophilic spinning fibers;

10. Cytokine or drug;

11. Spinning fibers of the transition layer;

A. Hydrophobic electrospun layer;

B. Hydrophilic electro-spun layer;

C. The transition layer.

The best embodiment of the invention

Now we will consider examples of carrying out the invention in more detail. Examples will be illustrated in the accompanying drawings, in which the same number represents the same elements. The examples described with reference to the drawings are only images that are intended to explain the invention and do not indicate limitations of the invention.

Now, along with Figures 1 to 8, we will now go on to describe the artificial dura mater according to the present invention and a method for its production.

An image of the electrospinning process for producing artificial dura mater is shown in Figure 1, the electrospinning spray apparatus 1 contains a polymer solution, the high voltage end of the high voltage power supply 3 is connected to the spray apparatus 1. The receiving device 4 has a cylindrical shape, and it can be moved to the left and / or right along the axis of the cylinder or the direction of the rod of the long axis of the cylinder. The movement of the receiving device 4 can be set using a computer program so that the formed electrospun layer will have the same thickness. In fact, the receiving device can be installed as a flat surface, and by moving left and right or forward and backward, you can ensure uniform reception. The receiving device 4 is connected to the low voltage end of the high voltage power supply 3, so that between the spray device 1 and the receiving device 4 there is a large voltage difference.

Before starting electrospinning, it is necessary to prepare an appropriate polymer solution for electrospinning.

You can choose a solution of a hydrophobic polymer for electrospinning, such a solution is made by dissolving the hydrophobic polymer in a solvent. Moreover, the aforementioned hydrophobic polymer includes, but is not limited to, a hydrophobic aliphatic polyester (including polylactic acid, polyglycolide, polycaprolactone and polyhydroxybutyrate), a polyester ether copolymer (for example, polydioxanone), a polyorthoester, polyurethane acid, for example, polyanhydride, for example ), polyphosphazene, polyamino acid and mixtures thereof.

Depending on the design, if a hydrophilic electro-spun layer is necessary after the hydrophobic electro-spun layer is finished, it is necessary to prepare a solution of a hydrophilic polymer for electrospinning. Moreover, the aforementioned hydrophilic polymer includes, but is not limited to, chondroitin sulfate, heparin, agar, Toyukan, algin, modified cellulose, sodium alginate, starch, cellulose, gelatin, fibrinogen, silk protein, elastin imitation polymer peptide, collagen, chitosan, modified hydrophilic polyurethane, polyethylene glycol, polymethyl methacrylate, polymethyl methacrylate, hydroxybutyrate co-hydroxyvalerate, polyhydroxybutyrate-co-hydroxyhexanoate (RNBHH), polyvinyl alcohol and polylactide. Based on the actual needs, several spraying apparatuses 1 can be installed in which the aforementioned hydrophobic polymer solution and the aforementioned hydrophilic polymer solution are respectively placed. Or, you can replace the solution in the spray apparatus 1 after the hydrophobic layer is completed.

The solvent for the electrospinning solution may be water or a volatile organic solvent, which includes, but is not limited to, methane acid, acetic acid, ethyl alcohol, acetone, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, hexafluoroisopropyl alcohol, trifluoroethyl alcohol, dichloromethane, trichloromethane, methyl dichloromethane , chloroform, dioxane, trifluoroethane and trifluoroacetic acid.

As soon as the electrospinning solution is ready, parameters must be set. After that, the power is turned on, and the electrospinning device is activated. Since the spinning fibers 2 are formed by spinning from a spray apparatus 1, the receiving device 4 will move in the established order so as to create a uniform structure of the electro-spun shell.

The parameters of the process of creating the aforementioned hydrophobic electrospun layer are set as follows: a micro-injection pump operating with a capacity of 0.1-5.0 ml / h, a high voltage generator operating with a voltage of 5-40 kV, and a feed distance of 5.0-30.0 cm The aforementioned hydrophobic electrospinning layer consists of fiber, the diameter of which can be controlled in the range from 50 to 1000 microns, and pores whose size is less than 3 microns.

The parameters of the process of creating the aforementioned hydrophilic electrospun layer are set as follows: a micro-injection pump operating with a capacity of 0.1-20.0 ml / h, a high voltage generator operating with a voltage of 10-45 kV, and a feed distance of 5.0-30.0 cm The aforementioned hydrophilic electro-spun layer consists of fiber, the diameter of which can be controlled in the range from 5 to 200 microns, and pores, the size of which is 20-200 microns.

In fact, the procedures described above can be repeated to create multilayer hydrophobic layers and / or multilayer hydrophilic layers, as shown in Figures 3 and 4.

The Figure 3 shows the artificial dura mater with three hydrophobic layers, the strength of which is similar to the strength of the dura mater of man. Since the layers are formed by hydrophobic materials, they are not suitable for the movement and binding of cells. However, the materials are safe, non-toxic and absorbed by the human body, they ensure the achievement of release.

The Figure 4 shows the artificial dura mater, consisting of two hydrophobic layers (A) and three hydrophilic layers (B). During transplantation of the dura mater into the brain, the hydrophobic release layers (A) are located close to the surface of the brain, while the hydrophilic layers (B) are located far from the surface of the brain, providing a thin nanofiber scaffold for adhesion, movement, growth and differentiation of cells. Since the hydrophilic layers are made of hydrophilic materials that provide good biocompatibility and have a larger pore size, which is suitable for the movement of stem cells and fibroblasts, which, therefore, is favorable for the growth of autologous dura mater.

Once the electro-spun layers are created, they must be dried in an oven or naturally, depending on the composition of the solution. If the solvent of the electrospinning solution is a volatile organic solvent such as hexafluoroisopropyl alcohol, the drying procedure can be neglected, since the solvent is completely volatilized, while the spinning fibers 2 are made by extrusion in the receiving device 4, as well as the voltage difference.

Since the hydrophobic layer is very different from the hydrophilic layer in terms of hydrophilicity, it is difficult to maintain structural stability in practice. To resolve such a problematic increase in hydrophilicity between these two layers, it is necessary to use a transition layer. The aforementioned transition layer has hydrophilicity, which gradually increases from the side closest to the aforementioned hydrophobic electrospun layer, to the side closest to the aforementioned hydrophilic electrospun layer. In fact, the electrospinning solution of the aforementioned transition layer may contain one or more polymers and an appropriate solvent, which are determined based on hydrophilicity requirements. Then, in the aforementioned manner, the solution is placed in a spray apparatus to obtain the aforementioned transition layer to the aforementioned hydrophilic layer. The parameters of the transition layer creation process are set as follows: a micro-injection pump operating with a capacity of 0.1-5.0 ml / h, a high voltage generator operating with a voltage of 5-40 kV and a feed distance of 5.0-30.0 cm.

An artificial dura mater with a transition layer is depicted in Figure 5. In this figure, the aforementioned hydrophobic layer consists of two layers, the aforementioned hydrophilic layer consists of three layers. Between the aforementioned hydrophobic layers (A) and the aforementioned hydrophilic layers (B), a transition layer is provided consisting of two layers (C), the layer closest to the aforementioned hydrophobic layers (A) has a weaker hydrophilicity than the layer closest to the aforementioned hydrophilic layers .

In addition, mixing electrospinning can be used to add a cytokine and / or drug to the artificial Dura. In particular, the cytokine and / or drug can be mixed with any one or more layers of the aforementioned hydrophobic, hydrophilic and transition layers. The cytokine and / or drug must be placed in an appropriate solution. After this, electrospinning is carried out and the cytokine and / or drug will be mixed with the spinning fibers 2 as the spinning fibers are formed. The shell structure will be formed on the receiving device 4. In addition, this procedure can be repeated. The cytokine and / or drug added each time may be the same or different. The resulting artificial dura mater is shown in Figure 6. In Figure 6A, the hydrophobic layer has a two-layer structure, and the hydrophilic layer has a three-layer structure. In Figure 6B, the hydrophobic spinning fiber 7 contains the drug 8, and the hydrophilic spinning fiber 9 is mixed with the cytokine 10.

Figure 7A has two transition layers C based on Figure 6. One of them closest to the aforementioned hydrophobic layer A has a weaker hydrophilicity than the layer closest to the hydrophilic layer B. From Figure 7 B, the hydrophobic dope fiber 7 is mixed with the drug 8 , the hydrophilic spinning fiber 9 is mixed with the cytokine 10, the spinning fibers of the two transition layers 11 are respectively mixed with the drug 8 and cytokine 9.

In addition, the invention provides a method that combines electrospinning and bioprinting to obtain artificial dura mater. The aforementioned bioprinting is the latest technology that has been recently developed and has been used in the biomedical field in recent years. This technology uses a special cell solution or biologically active solution, such as bio-ink, and, in accordance with the development, ensures printing precisely on the site of a specific substrate (called “bio-paper”), which can decompose in the human body. After printing, the bio-paper is stacked in a certain sequence. Using printing technology, bio-ink, consisting of cells and / or a cytokine, can be placed exactly in the required areas. Bio paper folded in a specific order forms a three-dimensional structure.

A specific implementation example is shown in Figure 2. Based on the device shown in Figure 1, a bioprinting head 5 is installed, such a head can be obtained by modifying an existing industrial inkjet printer, similarly to the method, for example, disclosed in US patent 7051654. The head contains a cytokine and / or drug. The printing method and the printable working position for printing can be set in advance through a computer program. Certain printing procedures can be based on modern technology.

In accordance with one embodiment of the invention, the cytokine and / or drug can be included in the hydrogel. The above hydrogel solution may be an aqueous solution of a polysaccharide polymer, a polypeptide polymer, or a synthetic hydrophilic polymer. The aforementioned polysaccharide polymer includes, but is not limited to, starch, cellulose, sodium alginate, hyaluronic acid, and chitosan. The aforementioned polypeptide polymer includes, but is not limited to, collagen, poly-L-lysine, and a lactic and glycolic acid copolymer (PLGA). The aforementioned synthetic hydrophilic polymer includes, but is not limited to, acrylic acid, polymethacrylic acid, polyacrylamide, and N-isopropyl acrylamide. Under normal conditions, the aforementioned hydrogel is liquid and turns into a gel at a certain temperature or under certain conditions. The hydrogel has good adhesion, which contributes to the uniform distribution of the cytokine and / or drug in the electrostatic spinning layer.

Hydrogel bioprinting procedures include the following steps: 1) Place the cytokine and / or drug prepared and mixed with liquid hydrogel in the bioprinting head 5. 2) After the formation of the electro-spun layer, ensure printing on the electro-spun layer using an inkjet printer according to the specified program; based on the choice of hydrogel, create the appropriate conditions for the hydrogel to quickly turn into a gel / become jelly-like, providing good adhesion, adhere the cytokine and / or drug to the electro-spun layers. 3) Establish a uniform and uniform distribution of bio-ink and form an artificial Dura mater, as shown in Figure 8A, where each of the hydrophobic layers is printed using a drug layer and each of the hydrophilic layers is printed using a cytokine layer. As shown in enlarged Figure 8B, the film prepared by bio-printing is different from the film prepared by mixing. The cytokine and / or drug is applied to the surface of the layers, which is formed from a hydrophobic spinning fiber 7 and / or a hydrophilic spinning fiber 9. When transplanting such a dura mater into the human body, the aforementioned cytokine and / or drug can be released uniformly. 4) If necessary, establish a concentrated distribution at specific points and print the aforementioned cytokine and / or the aforementioned drug in certain areas. When such a dura mater is transplanted into a human body, the aforementioned cytokine and / or go the aforementioned drug can be released mainly on the necessary specific areas.

The hardening of some hydrogels requires the use of a cross-linking agent. In this case, bio-printing using a hydrogel involves the following procedures: 1) Place a certain amount of cross-linking agent in the vessel 6. As soon as the electrospinning begins, the receiving device 4, moving along the axis or left-right, will begin to interact with the substance, and the formed electrospun layers will stick to cross-linking agent 2) Perform bioprinting as described above. When a liquid hydrogel interacts with a cross-linking agent on the electro-spun layers inside the head 5, the hydrogel quickly turns into a gel / becomes jelly-like and helps the cytokine and / or drug to adhere to the electro-spun layers. The choice of cross-linking agent depends on the type of hydrogel. For example, if the hydrogel is sodium alginate, the cross-linking agent is calcium chloride; if the hydrogel is fibrinogen, the agent is thrombin.

According to one embodiment of the invention, the aforementioned solution of the hydrophobic polymer can be hydrophobic poly (L-lactide) (PLLA) and ε-caprolactone dissolved in hexafluoroisopropyl alcohol or dichloromethane, the ratio of the above two polymers can be 50:50, 30:70 or 70: thirty. Like a copolymer, its number average molecular weight is 150000-500000. If mixing is required, a 0.01-3% antibiotic solution and / or 0.001-3% drug for hemostasis and release can be added to the hydrophobic solution. Together with a solution of poly (L-lactide) (PLLA) and ε-caprolactone we get the final solution.

According to another embodiment of the invention, the hydrophilic polymer solution may include hydrophilic polyurethane plus natural gelatin, chondroitin sulfate or polyethylene glycol as solvent (s). The mass ratio is 20-80: 80-20. The spinning solution is 3-15% of the total mass. When mixed in a solution of a hydrophilic polymer, you can add a solution of the main fibroblast growth factor and provide a cytokine concentration of 0.001-0.5%.

The drug added by mixing or bioprinting can be selected according to the actual situation: antibiotic or drug for hemostasis or release. During transplantation of the dura mater due to the removal of a tumor, drugs for chemotherapy of a brain tumor can be added.

An antibiotic includes, but is not limited to, cephalosporin, ampicillin, spiramycin, sulfonamides, and quinolones. The most preferred option is ceftriaxone sodium. In operations on the pia mater, it is often necessary to open the skull and intracranial infection often has a bacterial nature, mainly including Staphylococcus aureus, Streptococcus aureus, Streptococcus pneumonia, Escherichia coli, Salmonella and Pseudomonas aeruginosa. The most common virus is Staphylococcus aureus. According to clinical reports, ceftriaxone sodium provides the best therapeutic effect.

An antitumor agent includes, but is not limited to, nimustine, semustine, liposomal doxorubicin, dactinomycin D, and vincristine. Vincristine is the most preferred option.

A drug for hemostasis or release can accelerate wound healing and prevent adhesion. Such a medicine includes, but is not limited to, hemostasis factor (which helps the material become hemostasis), a collagen synthase inhibitor (e.g. tranilast and alamast, which inhibits collagen recovery), an anticoagulant (e.g. dicumarol, heparin, sodium and hirudin ), an anti-inflammatory drug (e.g., promethazine, dexamethesone, hydrocortisone, prednisone, ibuprofen and oxyphenbutazone), a calcium channel blocker (e.g., diltiazem hydrochloride, nifedipine and verapamil hydrochloride), a cell growth inhibitor (e.g. fluorouracil), hydrolase (e.g. hyaluronidase, streptokinase, urokinase, pepsin and tissue plasminogen activator (tPA)) and a redox preparation (e.g. methylene blue).

According to one embodiment of the invention, when mixed into the electrospinning solution, a cytokine and / or drug is added according to the formula: the main fibroblast growth factor is 0.001-0.05% by weight of the electrospinning solution, ampicillin is 3% by weight of the electrospinning solution, hemostasis factor is 0.001-0.05% of the mass of the solution for electrospinning. During surgery to restore the soft meninges due to a brain tumor, nimustine can be added, which is 0.01-5% by weight.

The resulting artificial dura mater must be washed, sterilized, packaged and deposited.

Example 1

Poly (L-lactide) (PLLA) and ε-caprolactone in a mass ratio of 50:50 and with a number average molecular weight of 260,000 are dissolved in a solvent of hexafluoroisopropyl alcohol to create a hydrophobic solution for electrospinning. Place the solution in an electrospinning spray device. The micro-injection pump operates with a capacity of 5 ml / h; the high voltage generator operates at a voltage of 30 kV and a feed distance of 20 cm. Fiber arrives to create a shell structure with an average diameter of 300 nm. After completion of the feeding process, the spinning device is closed.

The resulting artificial dura is washed five times with ethanol and distilled water. Then, after lyophilization, the dura mater is vacuum packed. After sterilization of 25 kGy of cobalt-60, the dura mater is stored at a temperature of minus 20 ° С.

Example 2

The method of obtaining the same as in Example 1.

For hydrophilic electrospinning solution, select poly oxalic acid and chondroitin sulfate in a mass ratio of 70:30 and with a mass fraction of spinning liquid of 9%.

An electrospinning device is driven and a hydrophilic electrospinning layer is formed on the hydrophobic layer already formed in Option 1. The feed distance is 11 cm, the voltage is 20 kV, and the resulting hydrophilic layer consists of fibers with an average diameter of 10 μm.

Rinse and store as in Embodiment 1.

Example 3

The hydrophobic electro-spun layer is manufactured in the same manner as in Embodiment 1.

The transition layer receives a polymer solution of polyurethane and hyaluronic acid in a mass ratio of 70:30 and has a mass fraction of 10%. Spinning is activated at a feed distance of 11 cm and a voltage of 20 kV. Fibers have an average diameter of 5 microns. A transition layer is created on the hydrophobic layer.

Then, a hydrophilic layer is made by spinning on the transition layer, as in Example 4. After this, spinning is stopped.

Rinse and store as in Example 1.

Example 4

(1) prepare a hydrophobic electro-spun layer: Select a hydrophobic polycaprolactone and a mixed solvent of chloroform or methyl alcohol in a 1: 1 ratio. Add ceftriaxone sodium at a concentration of 1%. Get a homogeneous solution.

Add the above solution to the electrostatic spinning apparatus and perform electrospinning using a micro-injection pump operating at a capacity of 0.8 ml / h, a high-voltage generator operating at a voltage of 12 kV and a feed distance of 15 cm. Obtain fibers in the sheath structure. The fibers of the hydrophobic electro-spun layer have a diameter of 600 nanometers.

Close the spinning device.

(2) prepare a hydrophilic electro-spun layer: Add a hydrophilic silk protein and natural gelatin in a ratio of 20-80: 80-20 and a mass fraction of spinning liquid of 9%.

Prepare a solution of the main fibroblast growth factor. Evenly mix the solution with the aforementioned electrospinning solution, the final concentration of the cytokine should be 0.001%, the feed distance should be 10 cm and the voltage should be 20 kV. Start electrospinning and create a hydrophilic layer on the already formed hydrophobic layer. The average diameter of the fibers from the hydrophilic layer is of the order of a micron.

Rinse and store as in Example 1.

Example 5

(1) prepare a hydrophobic electro-spun layer: select a hydrophobic polycaprolactone and a mixed solvent of chloroform and methyl alcohol in a 1: 1 ratio. Mix with vincristine at a concentration of 100 ng / ml and get a homogeneous solution.

Add the above solution to the electrospinning spray apparatus, adjust the micro-injection pump to 0.8 ml per hour, the voltage of the high voltage generator to 12 kV and the feed distance of the receiver to 15 cm, and obtain fibers in the sheath structure. The fibers of the hydrophobic electro-spun layer have a diameter of 600 nanometers.

Close the spinning device.

(2) prepare the transition layer: Select polyurethane and hyaluronic acid in a weight ratio of 70:30. The mass fraction of the dope should be 10%. Mix with ampicillin at a concentration of 3%. Prepared a homogeneous solution.

Start spinning. Fabricate a transition layer on an already formed hydrophobic layer by spinning.

The feed distance is 11 cm, the voltage is 20 kV and the average fiber diameter is 5 μm.

Close the spinning device.

(3) prepare a hydrophilic electro-spun layer: Add a hydrophilic silk protein and natural gelatin in a ratio of 20-80: 80-20 and a mass fraction of a spinning solution of 9%. Mix with a solution of ampicillin with a final concentration of 3%.

Turn on the spinning device, adjust the feed distance by 10 cm and spin the hydrophilic layer on the already formed transition layer. The average fiber diameter should be of the order of per micron.

Rinse and store as in Example 1.

Example 6

(1) prepare a hydrophobic electro-spun layer using bioprinting:

As a hydrophobic solution, hydrophobic poly (L-lactide) (PLLA) and ε-caprolactone, in a mass ratio of 50:50, the number average molecular weight of the copolymer dissolved in hexafluoroisopropyl alcohol, is 260,000.

As a cross-linking agent, a 0.1 mol.% Solution of calcium chloride is used.

A hydrogel containing a cytokine takes an alginate solution of hemostasis factor. The hemostasis factor has a concentration of 10 particles per million by weight in a cytokine alginate solution.

A solution with 0.1 mol.% Calcium chloride must be placed in a Petri dish with a diameter of 150 mm. Part of the receiving device, common to the spinning device and bioprinting, is placed in a Petri dish. When creating an electro-spun layer, the receiving device should interact with the solution in the Petri dish. The print head is installed under the electrospinning needle, which is located inside the spinning device, which provides fixed printing at a specific location with a hemostasis factor. The prepared cytokine alginate solution is placed in an inkjet printer cartridge. In this embodiment, the cartridge is HP51626A.

A hydrophobic electrospinning solution is placed in a spraying apparatus for spinning. After that, it is necessary to adjust the speed of the micro-injection pump to 5 ml / h, the voltage of the high-voltage generator to 30 kV and the feed distance of the receiving device to 20 cm. The fiber is placed in the shell structure. Spinning lasts 20 minutes before the spinning device is closed.

A hydrogel solution containing a cytokine is printed on the aforementioned nanobionic scaffold using an inkjet printer. As soon as the hydrogel hardens, a hydrophobic electro-spun layer with a cytokine is obtained using bioprinting.

(2) prepare a hydrophilic electro-spun layer using bioprinting:

Prepare a solution for electrospinning, a hydrogel solution with a drug, and a solution of a cross-linking agent.

Use polyglycol and chondroitin sulfate, as well as hydrophilic materials in a mass ratio of 70:30. The spinning solution has a mass fraction of 9%. A solution of cross-linking agent is 0.1 mol.% Solution of calcium chloride.

A hydrogel solution containing a cytokine takes an alginate solution with a major fibroblast growth factor. The above basic alginate solution has a major fibroblast growth factor at a concentration of 100 ppm.

The following parameters are set: set the micro-injection pump capacity to 0.8 ml / h, the voltage of the high-voltage generator to 20 kV and the feed distance of the receiving device to 11 cm. Other preparatory procedures are the same as in the aforementioned first stage. After placing the hydrophilic fiber in the shell structure using bioprinting, a hydrogel solution containing the main fibroblast growth factor is printed on a nanobionic scaffold. As soon as the hydrogel hardens, bioprinting produces a hydrophilic electro-spun layer with a cytokine.

Rinse and store as in Example 1.

Example 7

(1) preparing a hydrophobic electro-spun layer using bioprinting in the same manner as in Example 6.

(2) prepare the transition layer using bioprinting.

The electrospun liquid solution of the transition layer consists of polyurethane and hyaluronic acid in a mass ratio of 70:30. The mass fraction of the dope is 10%. The concentration of mixed ampicillin is 3%.

The solution of cross-linking agent is 0.1 mol.% Solution of calcium chloride.

A hydrogel solution with a cytokine is an alginate solution of hemostasis factor, the percentage by weight of hemostasis factor is 10 particles per million.

The parameters are configured as follows: the performance of the micro-injection pump is 4 milliliters / hour, the voltage of the high-voltage generator is 20 kV and the feed distance is 11 cm. Other procedures are the same as described above. The fiber is housed in a sheath structure. A hydrogel solution containing a cytokine will be printed on the transition layer. After the hydrogel has hardened, a hydrophobic electro-spun layer with a cytokine is obtained using bioprinting.

(3) A hydrophilic electro-spun layer is prepared using bioprinting in the same manner as in Example 6.

Rinse and store as in the above Example 1.

Experiment Example 1

The dura mater obtained from Example 1 is applied to dogs for conducting an experiment on animals, using a serial control experimental animal and restorative material of xenogenous meninges, applicable in medical practice. Three healthy dogs, male or female, are selected, the weight of which varies in the range from 10 to 15 kg, they are observed for 2-3 months. Dogs selected to participate in the experiment are under general anesthesia. Their skulls are opened on the left and right sides. Their dura mater is removed surgically, thus damaging the dura mater and injuring the brain tissue. Then, the dura mater obtained from Embodiment 1 is used to transplant the control experimental animal, respectively, in order to repair damaged dura mater from the left and right sides of the dog’s brain. After the operation, the dogs are fed as usual, and periodically observed. A sample is taken from the implanted area at the end of each observation period. At the end of the observation period after general anesthesia, the skulls of the dogs are opened by the aforementioned method after general anesthesia. Open and separate the outer surface of the reducing material. To obtain samples of these dogs are killed by intravenous air. Skulls are opened surgically. Take out the implants and surrounding tissue. The obtained samples are carefully examined, including their appearance, characteristics, their interaction with the environment, the protective shell, callus, and also the adhesion between their inner surface and the brain tissue. The sample is stored in a vessel and treated with a formaldehyde solution. Mark the vessel. After a week, remove local tissue; pour paraffin section and histological stain with hematoxylin and eosin.

A few days after the transplant, three dogs recover quickly, postoperative sutures heal well without any obvious discharge. Dogs eat and drink normally, while their activity is normal without any obvious impaired movement function. Three months after implantation, three dogs are sacrificed by intravenous air. After that, taking the surgical field as the center, the sample is taken one centimeter larger than the size of the operated site, and it includes regenerative material surrounding the dura mater and part of the brain tissue. After the sample has been taken, separate the skull from the meninges properly. It has been established that the pia mater, to which the dura mater was transplanted, is fully formed; the transplanted material was replaced by fibrous tissue, and firmly connected to the natural soft meninges without scars. The inner surface of the newly formed soft meninges at the transplant site is protected from adhesion to the brain tissue, while the surface of the corresponding brain tissue is smooth and protected from adhesion with the implant. At the same time, in the implanted area of the samples of the control experimental animals, the transplanted material did not begin to break down and there is some adhesion at the site of transplantation on the inner surface of the soft meninges.

Experiment Example 2

The dura mater prepared in Example 2 is now used in an experiment conducted on dogs. Five dogs weighing 15-20 kg, aged 1.5-2 years male or female. They are immersed in general anesthesia by intramuscular injection of ketamine. After anesthesia and shaving, animals are placed on the operating table in a position on the side of the abdomen. Disinfection with 2% iodine and 75% ethyl alcohol is carried out. The heads of animals are opened surgically in the longitudinal direction. A probe for extirpation is used to separate the periosteum from the skull, and the upper two sections of the skull are opened. A high-speed drill is used to open the skull. Two apertures are formed at the apical point of the skull, with two rectangular dura mater of 3 cm × 3 cm in size in the upper part of the head cut out with scissors. Ultimately, damage to the superior dura mater is created for subsequent transplantation of the artificial dura mater and control of the animal. On the open surface of the brain, electrocoagulation is used to create 6 damaged areas 1 mm × 1 mm in size. Then, the artificial dura mater made in Embodiment 2 is cut to fit the shape and size of the damaged areas of the dura and mounted on the damaged area. A hydrophobic layer is placed on the surface of the brain, and sutures for repairing damage to the dura of the dogs are made of 4/0 threads with an interval of 4 mm A surgical round needle and a 4/0 thread are used to connect the muscles. As a control experimental animal, a serial clinically acceptable restorative material of the dura mater of animal origin is used. After the operation, the dogs are fed as usual and periodically observed. Animals recover well and postoperative sutures heal quickly. Explicit cerebrospinal fluid or epilepsy was not detected. Dogs eat and drink as usual, their activity is normal without any obvious impairment of movement function, and they live to average life expectancy.

Eighteen months after the transplant, the dogs are sacrificed and a sample is taken on the surgical field, which includes the artificial Dura mater surrounding the Dura mater and part of the surrounding brain tissue. If you carefully examine the sample, you can see that the connection between the artificial dura mater and the natural dura mater is dense and smooth, without a clear border, well healed, in addition, only the thread is visible. Own dura mater does not show signs of hyperemia, hemorrhage or other rejection reactions. Moreover, the control results show that the material of the implant has not yet decomposed, and on the implanted area the inner surface of the soft meninges of the tissue adheres to the brain to some extent.

Experiment Example 3

The artificial dura mater obtained from Example 3 is now used in an experiment conducted on a New Zealand rabbit.

The experimental animals are exposed to the skull and create damage to the dura mater and brain tissue by surgery. Then, artificial dura mater is used to repair the damage. After surgery, rabbits are fed as usual and periodically observed. These animals are well restored. Eighteen months after the transplantation, the rabbits were euthanized and a sample was taken on the operative field. The sample includes artificial - dura mater surrounding the dura mater, and part of the surrounding brain tissue. If you carefully examine the sample, it can be seen that the epithelial cells cover the inner surface of the dura mater; fibrous tissue forms under the epithelium, fibroblast precursor cells rapidly grow, the number of collagen fibers increases. All this leads to the formation of new vascularized tissues, the growth of the natural dura mater, decomposition of the implant material, a decrease in the total mass of the implant and the formation of large capillary networks. At the border between old and new tissues, the reaction of inflammation of neutrophilic, lymphocytic, or any other cells was not detected, and a protective membrane is not formed. The arachnoid and brain tissue are normal.

Experiment Example 4

Dura obtained from Example 4 is now used in an experiment conducted on dogs, the method is the same as in Example experiment 2.

Fifteen months after transplantation, the dogs are euthanized and a sample is taken on the surgical site. The sample includes artificial dura mater surrounding the dura mater and part of the surrounding brain tissue. If you carefully examine the sample, you can see that the connection between the artificial dura mater and the natural dura mater is dense and smooth, without a clear border, completely healed, and only the thread is visible. The natural dura mater does not exhibit hyperemia, hemorrhage, or other rejection reactions.

Experiment Example 5

Dura obtained from Example 5 is now used in an experiment conducted on a New Zealand rabbit. As a recovering material, a serial clinically acceptable product of the dura mater of animal origin is used.

The method is the same as in Example of experiment 3. Fifteen months after transplantation, the rabbits are killed and a sample is taken on the surgical field. If you carefully examine the sample, it can be seen that the epithelial cells cover the inner surface of the dura mater; fibrous tissue forms under the epithelium, fibroblast precursor cells rapidly grow, and collagen fibers are produced. All this leads to the formation of new vascularized tissues, the growth of the natural dura mater, decomposition of the implant material, a decrease in the total mass of the implant and the formation of large capillary networks. At the border between old and new tissues, the reaction of inflammation of neutrophilic, lymphocytic, or any other cells was not detected, and a protective membrane is not formed. The arachnoid and brain tissue are normal. Moreover, the control results show that the material of the implant has not yet decomposed, and on the implanted area the inner surface of the soft meninges of the tissue adheres to the brain to some extent.

Experiment Example 6

Dura obtained from Example 6 is now used in an experiment conducted on dogs, the method is the same as in Example experiment 2.

Twelve months after the transplant, the dogs are euthanized and a sample is taken on the surgical site. The sample includes artificial dura mater surrounding the dura mater and part of the surrounding brain tissue. If you carefully examine the sample, you can see that the connection between the artificial dura mater and the natural dura mater is dense and smooth, without a clear border, completely healed, while only the thread is visible. The natural dura mater does not exhibit hyperemia, hemorrhage, or other rejection reactions.

Experiment Example 7

Dura obtained from Example 7 is now used in an experiment conducted on a New Zealand rabbit.

The method is the same as in Example of experiment 3. Twelve months after transplantation, the rabbits are killed and a sample is taken on the surgical field. If you carefully examine the sample, it can be seen that the epithelial cells cover the inner surface of the dura mater; fibrous tissue forms under the epithelium, fibroblast precursor cells rapidly grow, and the number of collagen fibers increases. All this leads to the formation of new vascularized tissues, the growth of the natural dura mater, decomposition of the implant material, a decrease in the total mass of the implant and the formation of large capillary networks. At the border between old and new tissues, the reaction of inflammation of neutrophilic, lymphocytic or any other cells is not detected, and a protective membrane is not formed. The arachnoid and brain tissue are normal.

Regardless of the presented and described embodiments of the present invention, a person skilled in the art can, without departing from the principles and theory, change, modify and replace these embodiments of the present invention according to the instructions in the scope of the invention and claims.

Claims (30)

1. Artificial dura mater made of electrospun layers using electrospinning technology, wherein the aforementioned electrospin layer consists of at least a hydrophobic electrospin layer, which is made of one or more hydrophobic polymers selected from polylactic acid and polycaprolactone. 2. The shell according to claim 1, characterized in that it includes at least a hydrophilic electro-spun layer located on the specified hydrophobic electro-spun layer. 3. The shell according to claim 2, characterized in that the aforementioned hydrophilic electrospun layer is made by a method comprising electrospinning using one or more hydrophobic polymers selected from the group consisting of chondroitin sulfate, heparin, agar, glucan, algin, modified cellulose, alginate sodium, starch, cellulose, gelatin, fibrinogen, silk protein, polymer-peptide imitating elastin, collagen, chitosan, modified chitosan, hydrophilic polyurethane, polyethylene glycol, polymethylmethac Ilatov, with hydroxybutyrate-hydroxyvalerate, RNVNNh (polyhydroxybutyrate-co-hydroxyhexanoate), polyvinyl alcohol, polylactide and mixtures thereof. 4. The shell according to claim 2, characterized in that it includes a transition layer located between the hydrophobic and hydrophilic electrospun layers. 5. The shell according to claim 4, characterized in that the aforementioned transition layer is made by a method comprising electrospinning using one or more hydrophobic polymers, while the aforementioned transition layer has hydrophilicity, which gradually increases from the side closest to the aforementioned hydrophobic electrospin layer the side closest to the aforementioned hydrophilic electrospun layer. 6. A shell according to any one of claims 3 or 5, characterized in that the aforementioned polymers are mixed with a cytokine and / or drug. 7. A shell according to any one of claims 1 or 6, characterized in that it further comprises a cytokine and / or drug, which adhere to the aforementioned hydrophobic electro-spun layer and / or the aforementioned hydrophilic electro-spun layer. 8. The shell according to claim 7, characterized in that the aforementioned cytokine is selected from the group consisting of interleukin, colony stimulating factor, tumor necrosis factor, platelet growth factor, main fibroblast growth factor, and combinations thereof. 9. The shell according to claim 7, characterized in that the aforementioned drug is one or more selected from the group consisting of antibiotics, hemostatic agents, release agents and anti-tumor drugs. 10. The shell according to claim 7, characterized in that the aforementioned cytokine and / or drug are introduced into the hydrogel. 11. The shell according to claim 7, characterized in that the aforementioned hydrogel consists of one or more substances selected from the group consisting of a polysaccharide polymer, a polypeptide polymer and a synthetic hydrophilic high molecular weight polymer. 12. The shell according to any one of claims 1, 2 or 4, characterized in that the aforementioned hydrophobic electro-spun layer consists of a fiber with a diameter of 50-1000 nm. 13. The shell according to item 12, characterized in that the aforementioned hydrophobic electro-spun layer consists of pores whose size is less than 3 microns. 14. The shell according to claim 2 or 4, characterized in that the aforementioned hydrophilic electro-spun layer consists of fiber with a diameter of 5-200 microns and a pore size of 20-200 microns. 15. A method of manufacturing an artificial dura mater, comprising the following steps:
a) dissolving a hydrophobic polymer in a solvent in order to obtain a hydrophobic solution for electrospinning, wherein the aforementioned hydrophobic polymer is selected from the group consisting of hydrophobic aliphatic polyester, polyester-ether copolymer, polyorthoester, polyurethane, polyanhydride, polyphosphazene, polyamino acids,
b) fabricating by electrospinning a hydrophobic electrospinning layer such as a film from the aforementioned hydrophobic electrospinning solution, thereby producing the aforementioned artificial dura mater. 16. The method according to clause 15, wherein the aforementioned hydrophobic aliphatic polyester is at least one substance selected from the group consisting of polylactic acid, polyglycolide, polycaprolactone and polyhydroxybutyrate. 17. The method according to clause 15, wherein the aforementioned polyester-ether copolymer is at least one substance selected from the group consisting of polydioxanone, a glycol / lactic acid copolymer and a polybutylene terephthalate / glycol copolymer. 18. The method according to p. 15, characterized in that the aforementioned hydrophobic electrospun layer consists of fibers with a diameter of 50-1000 nm. 19. The method according to p. 18, characterized in that the aforementioned hydrophobic electro-spun layer consists of pores whose size is less than 3 microns. 20. The method according to clause 15, characterized in that in step b) the aforementioned electrospinning is performed using a micro-injection pump operating with a capacity of 0.1-5.0 ml / h, a high voltage generator operating with a voltage of 5-40 kV, and with a feed distance of 5.0-30.0 cm. 21. The method according to p. 15, characterized in that it further includes creating a hydrophilic electrospin layer on the aforementioned hydrophobic electrospin layer, which includes the following steps:
a ') dissolving the hydrophilic polymer in a solvent in order to obtain a hydrophilic solution for electrospinning, wherein the aforementioned hydrophilic polymer is selected from the group consisting of chondroitin sulfate, heparin, agar, glucan, algin, modified cellulose, sodium alginate, starch, cellulose, gelatin, fibrinogen, silk protein, polymer-peptide imitating elastin, collagen, chitosan, modified chitosan, hydrophilic polyurethane, polyethylene glycol, polymethyl methacrylate, hydroxybutyrate co-hydroxyvalera ata, RNVNNh (polyhydroxybutyrate-co-hydroxyhexanoate), polyvinyl alcohol, polylactide and mixtures thereof;
b ') placing by electrospinning the aforementioned hydrophilic electrospinning solution on the aforementioned hydrophobic electrospinning layer to form the aforementioned hydrophilic electrospinning layer. 22. The method according to item 21, wherein the aforementioned hydrophilic electro-spun layer consists of fibers with a diameter of 5-200 microns and pores, the size of which is 20-200 microns. 23. The method according to item 21, characterized in that in step b ') the aforementioned electrospinning is performed using a micro-injection pump operating with a capacity of 0.1-20.0 ml / h, a high voltage generator operating with a voltage of 10-45 kV , and with a feed distance of 5.0-30.0 cm. 24. The method according to p. 15 or 21, characterized in that the aforementioned solvent is selected from the group consisting of methanoic acid, acetic acid, acetone, dimethylformamide, dimethylacetamide, dihydrofuran, dimethyl sulfoxide, hexafluoroisopropyl alcohol, trifluoroethyl alcohol, dichloromethane, methyl trichloromethane, trichloromethane ethyl alcohol, dioxane, trifluoroethane and mixtures thereof. 25. The method according to item 21, characterized in that it further includes, prior to the manufacture of the aforementioned hydrophilic electrospun layer, the creation of a transition layer by electrospinning the corresponding electrospun solutions consisting of one or more polymers between the aforementioned hydrophilic and hydrophobic layers, while the aforementioned transition layer has hydrophilicity , which gradually increases from the side closest to the above hydrophilic electrospun layer, to the side closest to the above hydrophobic electro-spun layer. 26. The method according to any of paragraphs.15, 21 or 25, characterized in that the aforementioned polymer solution for electrospinning also consists of a cytokine and / or drug. 27. The method according to p. 15 or 21, characterized in that it further includes the distribution by bioprinting of the cytokine and / or drug on the aforementioned hydrophobic electrospinning layer and / or the aforementioned hydrophilic electrospinning layer. 28. The method according to item 27, wherein the aforementioned bioprinting includes the following steps:
a '') mixing a hydrogel solution with the aforementioned cytokine and / or drug to create a solution; and
b '') printing the aforementioned solution on the aforementioned hydrophobic and / or hydrophilic electro-spun layers using bioprinting technology. 29. The method according to p. 28, characterized in that it further includes pre-processing the aforementioned hydrophobic and / or hydrophilic electrospun layers with a solution consisting of a cross-linking agent, to bioprinting. 30. The method according to p. 28, characterized in that the aforementioned hydrogel solution consists of a polysaccharide polymer, a polypeptide polymer, a synthetic hydrophilic high molecular weight polymer, or mixtures thereof.

Images